PROJECT SUMMARY The goal of this project is to understand the mechanisms responsible for activation of respiration in response to DNA damage. Because maintenance of genome stability is crucial for survival, cells have evolved a set of highly conserved mechanisms to sense and signal damaged DNA. These mechanisms are collectively referred to as the DNA damage checkpoint (DDC). In addition to DDC, eukaryotic cells also have DNA replication checkpoint (DRC) that specifically signals slowly progressing or arrested replication forks. Activation of DDC or DRC triggers transcriptional reprogramming of metabolism, required for coping with genotoxic or replicative stress. It is widely believed that DDC downregulates respiration to protect DNA from endogenous reactive oxygen species. Our preliminary data go against this dogma and show that DDC activates respiration to increase ATP production and elevate deoxyribonucleoside triphosphate (dNTP) levels, which are required for efficient DNA repair and cell survival upon DNA damage. The underlying mechanism involves DDC-induced downregulation of histone expression and altered chromatin structure, leading to increased transcription of respiratory genes. These findings reveal an unexpected connection between respiration and DDC, and indicate that the benefit of increased dNTP synthesis for cell survival offsets the deleterious effects of respiratory metabolism on DNA repair. The central hypothesis of this proposal, based on our preliminary data, is that activation of DDC or DRC induces transcriptional reprogramming, leading to increased expression of genes encoding enzymes of tricarboxylic acid cycle, electron transport chain, oxidative phosphorylation, and ATP synthesis. This hypothesis will be tested in two Aims that focus on the transcriptional mechanisms underlying the DDC- and DRC-induced respiration. In Aim 1, we will determine the mechanism of how DDC downregulates expression of histone genes. In Aim 2, we will determine the mechanism through which DRC upregulates respiration independently of histone gene expression. Experiments in both Aims will use yeast Saccharomyces cerevisiae as a model organism. Yeast is an ideal organism for the proposed studies, since it allows use of genetic, molecular, and biochemical tools, and is very appropriate for the involvement of undergraduate students, who will participate in all aspects of the study. Since defects in the repair of damaged DNA and resulting genome instability are associated with many cancers, analysis of the regulatory links between DDC, DRC and transcriptional reprogramming of metabolism will contribute to the identification of novel targets and approaches for cancer treatment. This project is innovative because it provides a completely novel perspective on the relationship between DNA damage and aerobic metabolism. This project will also provide an excellent research environment for motivated undergraduate students.